CROSS  INOCULATION  AS  INFLUENCING 
NITROGEN  FIXATION 


BY 

JAMES  GEORGE  KALLAS 


THESIS 


FOR  THE 

DEGREE  OF  BACHELOR  OF  SCIENCE 

IN 

AGRICULTURE 


COLLEGE  OF  AGRICULTURE 

UNIVERSITY  OF  ILLINOIS 


1922 


, 


UNIVERSITY  OF  ILLINOIS 


JUTlg § a 19  fr  fr. 


THIS  IS  TO  CERTIFY  THAT  THE  THESIS  PREPARED  UNDER  MY  SUPERVISION  BY 
J % . . .Q.Sfi  £g£ . 1 i&S. 


entitled £ JTQg.^.  .EA&ftkUl  A 


IS  APPROVED  BY  ME  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF £aS  .fc e.l.G  Jf. . .Q  £ . . 5.9. i .ett« £ . . .itt . . £& X'iSU  1 .t.UX.ft 


HEAD  OF  DEPARTMENT  OF 


Mr.Q  &QM. 


TABLE  OF  CONTENTS 


Introduction.  1 

Review  of  Literature  2 

The  Proo  lexa  2 

Experiment  I 3 

Experiment  II  8 

Part  II  12 

Review  of  Literature  12 

Experiment  III  15 

SvuSi&ry  IS 

Acknowledgment  19 

Bibliography  * 20 


TEE  INFLUENCE  OF 


CROSS- INOCULATION  ON 


NITHOGEN  FIXATION . 

INTBODUCTION 

llany  cross- inoculations  are  recognized  among  the  bacteria  common  to 
various  legumes.  The  most  complete  and  extensive  work  dealing  :ith  the  proof 
of  cross-inoculations  has  resulted  from  the  v/ork  of  Burrill  and  Hansen,  and 
Whiting  and  Hansen,  and  whiting  and  others.  The  groups  established  by  these 
workers  have  been  verified  by  other  workers.  While  the  groups  established 
contain  the  most  important  legua.es  now  under  cultivation,  it  is  clearly  recog- 
nized that  many  other  crosses  yet  undetermined  probably  occur. 

It  is  conceded  that  cross-inoculation  results  in  the  production  cf 
nodules  and  in  an  increased  growth  of  the  plants,  together  with  an  increase  in 
the  nitrogen  content.  The  fundamental  relationships  responsible  for  an  or- 
ganism adapting  itself  to  legu.es  of  widely  different  characteristics  have  not 
been  discovered.  Whether  the  morphology  of  the  root  structure  of  the  legumes 
in  a given  group  plays  a part  of  this  adaptation  is,  cf  course,  not  known  but 
snould  be  carefully  considered.  The  chemical  nature  of  the  cell  sap  cf  the 
plants  within  a given  group  _.lso  offers  a possible  line  of  investigation  which 
may  assist  in  clearing  -up  what  appear  to  be  peculiar  relationships  of  the  nodui 
bacteria.  It  might  be  assumed  that  a given  cheudcal  substance,  such  as  an 
alkaloid,  existed  in  ail  the  species  contained  in  a given  group.  Another  con- 
sideration which  should  be  carefully  eliminated  deals  with  the  production  cf 
specific  enzymes  by  the  organisms  associated  with  the  various  groups. 

There  are  two  distinct  organises  responsible  for  the  inoculation  cf 
legumes  from  the  standpoint  of  the  bacteriological  classification.  One  is  a 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/crossinoculationOOkall 


typical  peritrichic  ‘bacillus  and  of  this  form  there  are  probably  numerous 
varieties,  such  as  these  inoculating  red  clover,  alfalfa,  garden  peas,  and 
garden  beans.  The  other  organise  is  a g seudononas  possessing  a flagellum, 
net,  however,  typically  attached.  The  attachment  of  the  flagellum  has  been 
described  as  on  the  corner  instead  of  at  the  pole.  Two  totally  distinct 
varieties  of  the  pseudomonas  are  definitely  recognized,  that  of  the  cowpeas 
and  that  of  the  soybeans.  In  all  probability,  there  are  others,  although  they 
have  not  yet  been  found.  It  was  not  until  puite  recently  that  pure  cultures 
of  these  organisms  were  obtained  because  of  a lack  of  suitable  methods  for 
testing  out  the  purity  of  the  organism.  The  work  reported  by  Uniting  and  Hansen 
was  conducted  with  cultures,  the  purity  of  which  was  absolutely  proved  before 
and  after  making  the  crosses  reported. 

The  investigation  reported  in  this  paper  deals  with  the  efficiency  of 
the  process  cross-inoculation,  from  the  standpoint  of  the  i~-  ediate  adaptation 
of  an  organism  in  a given  group  to  any  -ember  of  that  group  upon  which  it  has 
not  recently  produced  nodules.  A specific  example  would  be  the  inoculation 
of  cowpeas  with  bacteria  previously  isolated  from,  peanuts,  in  comparison  with 
bacteria  previously  isolated  fro-  the  co\*p  sa  itself.  In  other  -words,  would 
there  be  an  advantage  in  always  using  for  inoculation  bacteria  from  like  legumes 
instead  of  bacteria  from  any  legumes  within  an  established  group . 

REVIEW  0?  LITERATURE 

A survey  of  the  literature  failed  to  reveal  any  work  dealing  with  the 
problem  under  study. 

THE  PROBLEM 

The  first  part  of  this  *rcrk  was  planned  to  measure  the  nitrogen  content 
of  legumes  inoculated  -with  bacteria  common  to  the  legume  to  be  used  in  the 
study  and  with  other  bacteria  producing  nodules  on  the  same  legume.  In  this 

roanner,  the  differences  in  nitrogen  content  in  the  presence  cf  nodule  bacteria 


. 


I 


-7- 


recently  from  the  legume  under  study  and  in  the  _resence  of  nodule  bacteria 
from  widely  different  legumes  might  be  obtained. 

EXPSEII4ENT  I 

Cross-Inoculation  of  Cowp eas  as  Influencing  Fixation. 

Cowpeas  were  selected  as  the  legume  to  be  inoculated  with  peanut,  par- 
tridge pea,  beggarweed,  and  lima  bean  a ong  the  nodule  bacteria,  and  in  addition 
with  Eadiobaoter  and  Azotobacter.  The  differences  that  night  develop  in  the 
growth  and  nitrogen  content  of  cowpea  plants  with  these  various  sources  of  inoc- 
ulation were  to  be  used  as  the  criteria  for  judging  the  efficiency  of  cross- 
inoculation  vs.  inoculation  with  bacteria  recently  from  the  cowpea. 

Preparation  of  Sand.  The  s.-nd  used  was  a high  =rude  clean  white  sand. 

It  was  sterilized  in  1-gallon  earthenware  jars  at  350°  F.  for  several  hours. 
Tests  for  nitrate  failed  to  show  its  presence  in  the  sand  grains.  After 
sterilization,  the  jars  were  allowed  to  aerate  for  48  hours.  Approximately  5 
gra  s of  nitrogen- free,  re-precipitated  CaCQ-  was  added  to  each  jar  and  thor- 
oughly mixed  with  a sterile  spatula.  The  moisture  content  of  the  sand  was 
established  at  12  percent  and  maintained  at  about  that  percent  throughout. 

Seeds  ? lant ed . Seeds  possessing  unbroken  seed  coats  were  selected.  In 
the  first  experiment,  the  cowpeas  were  selected  from  a large  sample;  in  the 
second  experiment,  the  sample  of  garden  x sas  was  somewhat  smaller.  The  seeds 
were  sterilized  in  1 to  500  mercuric  chloride  for  10  minutes.  They  were  washed 
with  distilled  water  several  times  and  they  were  allowed  to  dry  and  were  then 
placed  in  a sterile  jar  and  kept  ready  to  use. 

Plant  Food  Applied.  The  Jars  were  ^.reviled  with  all  plant  food  with 
the  exception  of  nitrogen.  The  plant  food  was  prepared  fro-  - pure  nitrogen-free 
chemicals  and  a - onia-free  distilled  water. 

Solution  No,  1.  Phosphorus  and  potassium:  Dissolve  28  grams  of  K__P?04 


- Il- 


ia 2p00  c.  c.  au-vonia-f res  wat er.  Use  10  o.  c.  per  jar. 

Solution  No.  2.  Magnesite.:  Dissolve  20  grams  cf  '-IgSQU  in  2500  c.  c. 

ammonia-free  water.  Use  10  c.  c.  per  jar. 

Solution  No.  3*  Iron:  Dissolve  0.1  gram  of  FeCl-?  in  250  c.  0.  amaonia- 

free  water.  Use  1 c.  o.  per  jar. 

The  solutions  were  applied  as  follows:  10  c.  0.  K9HPO4  -f-  10  c.  c. 

figSO^  4-  1 c.  c.  FeCl - made  up  to  a total  volume  of  100  c.  c.  with  aramonia-f 


water  for  each  jar.  The  first  application  was  :.-ade  at  the  time  of  planting, 
the  second,  two  weeks  later,  and  the  subsequent  applications  at  intervals  of 
one  week. 

Source  of  Inoculation.  The  organises  used  for  the  experiments  in  this 
work  were  transferred  cultures  froi'  Dr.  A.  L.  Whiting's  pure  stock  cultures. 
The  organisms  were  & rown  in  standard  :annite  agar  edia  the  composition  of 
which  is  as  follows: 


Agar  (powdered) 

15  g 

ra^-s 

Uannite 

10 

tf 

DNO- 

0.2 

it 

K^HPOji 

0.2 

1? 

MgS04 

0.2 

11 

NaCl 

A A 

• L 

it 

CaSOh 

0.1 

tt 

FeClj 

3 drops  of  10fo 

so lution 

CaCOj 

■ excess . 

Water 

1000  c . c . 

The  inoculation  was  applied  as  a suspension  over  the  seeds  at  the  time 
of  planting.  The  seeds  in  Exp eri  ent  1 were  planted  October  13 » 1921.  There 
were  12  seeds  planted  in  each  jar.  The  kinds  cf  inoculation  as  well  as  the 


-5- 


ana  lysis  of  the  plants  is  shown  in  Table  1. 

The  plants  were  treated  for  mildew  three  different  tines  with  a sulfur 
compound  which  contained  no  nitrogen.  Some  of  the  plants  were  effected  more 
than  others  and  their  growth  was  somewhat  stunted.  The  plants  grown  in  the 
jars  inoculated  with  Radio barter  and  Aaotobacter  organisms  began  to  turn  yellow' 
five  v/eeks  after  planting  and  finally  all  of  the  plants  grown  in  these  jars 
turned  yellow  and  growth  ceased.  The  plants  in  the  ether  jars  were  larger  and 
had  larger  leaf  surface  a*id  continued  to  grow.  The  only  difference  observed 
between  the  jars  inoculated  with  the  nodule  organisms  was  found  in  the  two 
jars  inoculated  with  peanut  bacteria.  The  plants  in  these  jars  were  more 
vigorous  and  a little  larger  than  the  ethers.  This,  however,  night  have  been 
due  to  the  fact  that  they  were  not  attacked  by  ildew  as  badly  as  some  of  the 
other  jars.  The  control  jars  began  to  turn  yellow  at  the  time  the  Azote  one  ter 
and  Radi obac ter  jars  did,  but  they  were  contaminated  later  and  began  to  turn 
green  again. 

The  plants  were  harvested  December  l4,  1321.  The  check  jars  were  con- 
taminated, nodules  were  found  in  their  roots.  The  jars  inoculated  with 
Azotcbacter  were  also  contaminated  and  began  to  turn  yellow  at  the  time  of 
harvest.  They  were  consequently  omitted  from  the  table  of  data.  There  were 
a f ew  nodules  found  in  their  roots.  There  were  ohly  two  nodules  found  in  the 
jars  inoculated  with  Radiobacter  and  these  were  used  as  checks.  The  complete 
plants  were  harvested  and  ost  of  the  sand  was  removed  by  wasning  the  roots. 

It  was  impossible,  however,  to  remove  all  the  srnd  without  losing  part  of  the 
roots,  and  therefore  some  of  the  sand  remained  on  the  roots.  The  plants  from 
each  jar  were  placed  in  separate  cheese-cloth  bags  and  were  carefully  labeled. 
The  bags  containing  the  plants  were  placed  in  an  electric  oven  and  the  plants 
dried  at  C.  The  plants  were  weighed  and  their  dry  weight  was  recorded  . 


■ 


■ 


. 


' 


. 


' 

* 


_S- 

The  seed  coats  and  cotyledons  that  were  collected  during  the  period  of  growth 
of  the  plants  were  added  to  their  respective  jar  of  dry  plants  and  were  in- 
cluded in  the  total  dry  weight. 

Method  of  Analysis.  The  total  plants  from  each  jar  were  digested  with 

35  c.  c.  concentrated  nitrogen-f ree  5 ijrams  of  ITa^SgO^  0.2  grams  of 

CaSOd  in  800  c.  c.  Kjeldahl  flasks  until  the  solution  was  clear  and  no  trace 

of  undigested  organic  matter  was  present.  The  sand  that  remained  on  the  roots 

of  the  plants  was  removed  during  the  decantation  of  the  digested  solution  and 

was  placed  in  separate  dishes.  The  sand  was  dried  and  weighed  ana  the  weights 

were  subtracted  fror  their  respective  original  dry  weights  of  the  cowpeas.  This 

gave  the  exact  total  dry  weight  of  the  plants.  The  digested  solution  was  ua&e 

up  to  500  c.  c.  in  500  c.  c.  measuring  flasks.  One  tenth,  or  ^0  c.  c.,  of  this 

solution  was  taken  for  analysis.  This  portion  was  poured  into  Kjeldahl  flasks 

and  the  solution  was  made  up  to  2p0  c.  c.  with  ammonia- free  water.  Excess 

concentrated  alkali  (KOH)  solution  was  addeu  arid  a few  granules  of  zinc.  These 

solutions  were  distilled  over  as  IJH-j  in  1 mg.  HC1  solution.  The  excess  of  acid 

J 

was  neutralized  ty  0.5  mg.  alkali  (.KOH)  arid  the  total  amount  of  nitrogen  was 
thus  determined.  The  results  of  the  analysis  are  shown  in  Table  1. 

Experiment  1 was  partly  repeated  (April  11  - May  31,  1322)  in  one  part 
glass  jars  with  the  following  inoculation:  Lira  bean,  cowpea,  beggarweed, 

Azotobacter  and  Had lob uo ter  organisms.  The  plants  in  the  check  jars  and  those 
in  the  jars  inoculated  with  Azotobacter  arid  Kacliobactar  organisms  began  to  turn 
yellow  on  the  sixth  week  and  a few  days  later,  most  of  the  leaves  dropped.  The 
other  jars  remained  green  and  grew  .uch  taller  than  the  others.  There  was  no 
difference  in  the  ether  jars  as  far  as  growth  was  concerned.  Time  aid  not  per- 
mit for  the  analysis  of  these  plants,  but  they  confirm  the  first  part  of  the 
experiment  as  far  as  behavior  of  the  plants  during  growth  is  concerned. 


-s- 


Eesults  of  Experiment  T 

The  results  of  this  experiment  show  that  the  plants  inoculated  with 
peanut  bacteria  gave  the  highest  amount  of  nitrogen  fixed.  Those  inoculated 
with  ccwpea,  beggarweed,  and  partridge  pea  (Cassia)  run  ail  of  about  the  sa  .e 
nitrogen  content,  and  those  inoculated  with  Rndiobacter  served  as  checks.  In 
the  latter  case,  it  shows  that  where  the  organism  did  net  lead  tc  nodule  forma- 
tion, the  plant 8 did  not  derive  any  benefit. 

The  larger  fixation  found  with  the  peanut  bacteria  ay  have  been  due  to 
these  plants  being  less  injured  by  the  mildew.  It  is  significant,  however,  that 
they  should  be  as  good  or  better  than  plants  inoculated  with  cowpea  bacteria. 

EXPERIMENT  II. 

Cross-Inoculation  cf  Garden  Peas  (Alaska)  as 
Influencing  Fixation. 

Tr.is  experiment  was  conducted  tc  study  the  effect  of  cross- inoculation 
of  garden  pea  with  vetch  organisms,  also  the  efficiency  of  different  pea  culture 

The  procedure  .and  experimental  methods  were  the  sa  .e  as  that  in  the 
exp eriment  I.  The  seeds  were  planted  October  ZB,  1321.  Twelve  seeds  were 
planted  in  each  jar.  The  plant  food  was  applied  in  the  same  manner  as  in 
experiment  1.  As  far  as  could  bs  observed,  there  was  no  difference  in  growth. 
The  height  of  the  plants  varied  somewhat,  but  that  was  also  true  of  plants  in 
the  same  jars.  The  plants  were  harvested  December  22,  1021.  The  checks  were 
contaminated,  but  since  there  were  no  checks  necessary  they  did  net  alter  the 
results  of  the  experiment.  1/Thile  harvesting,  great  care  was  taken  in  observing 
the  root  development,  the  number  of  pods  formed,  and  the  average  height  of  the 
plants.  There  was  hardly  any  difference  in  root  development  in  all  cf  the  jars. 
There  was  some  difference  in  the  number  cf  pods  developed,  however,  the  number 


■ 


. 


. 


' 


. 


-Q- 

j 

of  pods  are  recorded  for  each  jar  in  Table  2. 

Tne  process  cf  analysis  was  the  sa.-e  as  that  employed  in  the  experiment 
with  the  cowpeas,  vith  the  exception  that  the  plants  were  dried  in  squall  tin 
boxes.  There  were  20  jars  inoculated.  The  cultures  used  were  transfers  from 
Dr.  A.  L.  Whiting's  stock  cultures,  two  were  obtained  from  Dr.  E.  B.  Free  of 
the  Wisconsin  Station.  There  were  l4  Jars  representing  7 cultures  of  garden 
pea  bacteria  of  different  cultural  history,  and  4 jars  of  vetch  representing 
two  cultures  of  widely  different  cultural  history.  The  inoculation  was  rather 
heavy.  One  bottle  was  used  for  each  jar  and  the  cultures  were  cf  the  sane  age. 
The  treatments,  as  well  as  the  analysis  cf  the  plants  and  other  data,  is  given 
in  Table  2. 

Results  of  Experiment  II 

While  there  is  a difference  in  the  average  total  nitrogen  present  in 
the  plants  that  were  inoculated  with  the  two  pea  and  vetch  cultures  in  favor  of 
the  vetch  organise,  it  cannot  be  said  that  this  would  have  been  true  had  there 
been  the  same  number  of  jars  inoculated  in  both  cases.  However,  of  the  jars 
inoculated  with  garden  pea  bacteria,  four  might  be  taken,  such  as  23,  jO, 
and  40,  the  average  of  which  would  almost  be  tne  sa.  e as  the  four  vetch  jars. 

In  a problem  of  this  nature,  there  are  other  factors  that  enter  in  the 
results.  One  of  these  is  the  age  of  the  cultures.  Cultures  of  different  age 
will  have  different  effects  upon  the  plants.  The  condition  of  tne  growth  of 
the  plant  at  the  time  the  organism  is  at  its  highest  efficiency  would  be  another 
factor.  In  some  cases,  a plant  may  not  be  in  condition  to  use  all  of  the  nitro- 
gen produced  by  the  organism.  One  or  all  of  the  factors  may  be  ^.resent  in  some 
cases  and  therefore  give  results  which  would  lean  one  to  believe  that  one 
organism  ha 3 a better  effect  than  another  organism  in  cross- inoculation. 

It  would  require  mere  data  in  order  to  draw  conclusions  of  a reliable 


' 

. 


. - 


-10- 


nature,  but  the  oross-inoculat icn  in  this  ex_.eri.ient  proved  as  good  or  better 
than  the  inoculation  cf  peas  with  pea  bacteria. 


-12- 

PART  IT 

Nitrogen  Assimilation  "by  Non-legumes  Through  tae  Action  of 
Nodules  and  Free  Living  Nitrogen  Fixing  Bacteria. 

Experiment  3 was  planned  to  determine  whether  oats  could  benefit  hy  the 
presence  of  nodule  and  free  living  nitrogen- f ixing  bacteria.  Tne  import  nee  of 
the  problem  is  at  once  apparent  for  the  greatest  problem  in  American  agriculture 
would  be  solved  if  the  cereal  crops  c.uld  obtain  nitrogen  sufficient  for  their 
growth  from  nitrogen-f ixing  bacteria  living  in  the  soil. 

REVIEW  OF  THE  LITERATURE 

The  literature  dealing  with  nitrogen  assimilation  by  non- legumes  through 
the  presence  of  nitrogen-fixing  bacteria  is  more  extensive  than  that  dealing 
with  the  chemical  efficiency  of  cross- inoculation  vs.  inoculation  with  bacteria 
common  to  a given  legume. 

T vc  decidedly  different  views  have  been  ^ resented  regarding  the  conditions 
that  should  exist  for  the  non-legume  to  benefit  by  the  presence  of  the  nitrogen- 
fixing  bacteria.  These  views  are  very  simply  stated  as  follows:  (l)  That 

nodules  should  be  produced  on  the  roots  of  plants  in  order  for  a benefit  to  'be 
derived,  ( > ) that  nodule  production  is  entirely  unessential  in  order  to  p*er  it 
a non- legume  to  ass iaai late  the  nitrogen  fixed  by  the  bacteria. 

The  investigations  of  Schneider,  Burrill  and  Hansen,  Stutzer,  Burri  end 
Maul,  Grosbusch  and  Lee;  eraaann,  were  all  concerned  with  the  thought  that  nodule 
production  was  essential  for  success.  They  were  supporters  of  the  first  view. 

It  should  be  emphasized  here,  however,  that  so-**e  of  these  workers,  while  failing 
to  produce  nodules,  did  however  record  observations  indicating  that  in  their 
judgment  the  growth  of  the  non- legumes  was  beneficially  influenced  by  the 
presence  of  some  of  the  bacteria. 

Schneider,  working  under  the  late  Dr.  Burrill  at  this  station  in  1233, 


. 


- 13- 


cult  i vat ed  nodule  bacteria  froi  masse  lus  Vnlg.mris  upon  bean  extract  agar,  then 
upon  a fixture  of  bean  extract  and  corn  extract  agar,  and  finally  upon  pure  corn 
root  extract  agar.  Transfers  were  made  every  sixth  day.  Aft-r  the  cultures 
had  grown  for  a no  nth  up  on  the  pure  corn  root  extract,  they  "ere  ap-.  lied  upon 
germinating  seed  of  corn  aaxd  oats.  Though  the  inoculated  corn  plants  produced 
no  nodules,  Schneider  claimed  that  they  were  more  thrifty  than  the  unincculated 
plants.  He  described  and  figured  the  infection  of  some  of  the  root  hair  cells 
as  well  as  some  of  the  epidermal  and  parenchi.nl  cells.  No  effect  was  noticed 
upon  oats. 

Burrill  and  Hansen  took  up  the  problem  where  Schneider  left  it.  They 
attempted  to  inoculate  tomato  seedlings  vith  PseudO'-enas  Iladi ci cola  and  sweet 
clover  organises.  They  found  no  abnormal  conditions  in  their  roots,  but  the 
plants  that  were  inoculated  were  mere  thrifty  in  every  case.  Other  cases  in 
this  direction  have  been  reported.  Stutzer,  Burri  and  haul  inoculated  mustard 
plants  with  nodule  bacteria  which  had  gradually  become  accustomed  to  a custard 
plant  medivna,  but  without  success.  Grosbusch  experimented  with  C-ram  inae  but  his 
results  were  negative. 

Lemuermann  studied  the  difference  in  nutrition  between  the  Leguminosae 
and  the  Ora-:- in.ee.  He  believed  that  the  reason  for  the  existence  of  bacterial 
symbiosis  in  the  Leguminosae  and  not  in  the  Ora,  mans  are,  namely,  the  smaller 
transpiration  current,  the  higher  acidity  cf  the  root  sap,  and  the  greater  root 
develop/  ent  of  the  former  as  compared  with  the  latter. 

The  second  view  is  supported  by  the  striking  results  reported  by  H . V. 
Joshi,  Agricultural  Research  Institute,  Pusa,  India.  His  conception  of  the 
benefit  derived  by  non- legumes  is  based  upon  the  fact  that  nitrogen  fixation 
as  such  is  a function  cf  certain  bacteria  and  it  will  proceed  regardless  of  the 
nature  of  the  plant  present.  Any  plant  requiring  nitrogen  should  be  able  to 
assimilate  the  product  of  these  bacteria. 


. 


TA’SL'S  III  - Pronin r results  obtained  'y  •;.  y.  JohM  with  corn,  oats,  and  wheat. 


-15" 


He  conceived  the  idee,  while  he  was  conducting  a cross- inoculation  experi- 
ment with  peas,  math,  and  grab*  inoculated  with  two  strains  of  cowpea  organises 
and  arhar,  dhaincha,  and  gokarn  c rgani sms . When  he  exa;  i ned  the  roots  of  the 
plants  tnat  were  inoculated,  although  there  were  no  nodules  present,  still  their 
roots  appeared  much  ~ore  vigorous  than  those  of  the  uninoculated,  the  inoculation 
of  the  organ  is..;  appearing  to  exercise  a stimulating  effect.  He  then  suggested 
t nat  nitrogen  night  he  fixed  in  the  sand  independent  to  the  plant.  Upon  this 
he  based  his  hypothetical  assumption  that  the  nitrogen  thus  fixed  must  he  equally 
available  to  any  kind  of  ^lant,  whether  belonging  to  legnminc sae  cr  not.  Also 
if  one  kind  of  nitrogen-fixing  organism,  could  bring  about  increased  growth, 
another  organism*  like  Azotubacter  ay  be  able  to  show  the  e;ae  effect.  The  re- 
sults of  his  experiments  with  corn,  oats,  and  wheat,  besides  the  legumes,  show 
marked  differences  in  total  nitrogen.  The  difference  of  growth  is  also  noticed 
in  the  photographs  which  accompany  his  work. 

EXPERIMENT  III. 

Oats  Grown  in  the  Presence  of  Nodule  and  Free-Living 
N i t ro g en- Fixing  Bacteria. 

The  procedure  of  this  experiment  was  the  same  as  in  the  previous  experi- 
ments, The  amount  and  rate  of  giant  food  was  the  sane  as  in  experiments  I and 
£ with  the  addition  of  two  applications  of  1C  c.  c.  of  . annite  to  jars  44  to 

?4  inclusive,  and  CaClIO-Tp  43^0  v/as  ad  led  to  jars  55  and  ^6.  The  aannite  solution 

✓ 

was  made  up  by  adding  5 grams  of  ..annite  in  200  c.  c.  of  nitrogen-free  distilled 
water.  The  oats  was  planted  ilarch  23>  1922.  There  were  15  seeds  planted  in 
each  jar.  There  were  no  differences  noticed  on  the  plants  until  April  4.  At 
this  time  the  jars  treated  with  Ca(NO*)o  were  best  and  jars  47  and  48  were  a 
trifle  better  than  the  rest.  The  other  jars  vere  about  the  sane.  From  this 
time  on,  jars  42  to  54  inclusive  turned  yellow  and  remained  so.  The  stems  of 


-lo- 

the  plants  in  these  jars  were  very  thin  in  comparison  with  the  plants  in  jars 
55  and  About  May  3»  some  of  the  plants  on  the  check  jars  and  also  in  the 

jars  treated  with  bacteria  started  to  seed.  On  ’■'ay  S,  all  of  the  plants  treated 
with  organisms  and  the  control  jars  seeded.  Jars  55  and  56  received  70  c.  c. 
of  Ca(l!0-)2  each  or  equivalent  to  41.51  H.  A picture  of  the  plants  was 
taken  May  11,  1922.  The  total  plants  were  harvested  May  16,  1922. 

The  method  of  analysis  was  the  same  as  that  employed  in  experiments  1 and 
2 with  the  exception  that  the  digested  solution  was  made  up  to  200  c.  c.  and 
one-half  of  the  solution  was  taken  for  analysis.  The  results  of  the  analysis 
are  shown  in  Table  3. 

Results  of  Experiment  III 

The  results  of  the  total  nitrogen  determinations  indicate  in  eight  out  of 
ten  cases,  no  gain  whatever  to  the  oats  for  the  nitrogen  that  may  have  been 
fixed  by  the  bacteria.  In  the  case  of  jar  numbers  4?  and  52,  a larger  increase 
is  evident,  but  unfortunately  the  number  of  plants  was  not  definitely  known  and 
therefore  it  cannot  be  said  it  was  a gain  due  to  the  presence  of  the  bacteria  or 
to  increased  number  of  plants.  Judging  from  the  percent  of  nitrogen,  it  might 
easily  be  ascribed  to  on  increased  number  of  plants. 

If  oats  could  benefit  from  the  presence  of  these  organisms,  there  should 
have  been  more  nitrogen  contained  in  she  eight  jars  than  found  in  the  checks. 


. . 


. I 


p II I I I 

. 


-17- 


table  V . 

Dry  weights  and  total  nitrogen  content  of  oats  growing 
in  the  presence  of  Badiobacter,  Azotobacter,  and 
the  bacteria  cf  sweet  clover,  garden  peas.,  aad 
co wg  eas . 


Jar 

numb  or 

Treatment 

Cult -tors 
number 

’eight 

cf 

1 lants 

Dry 

weight 
in  gins. 

Total  N 
ter 

. J&r  _ _ 

43 

Check 

C*£S  • 

7 O 

2.15C 

15.4 

44 

tt 

3^ 

£.080 

15.6 

43 

Badiobacter 

1 %~r 

30 

1.550 

14.6 

46 

V 

140 

£3 

2.300 

13.2 

4? 

Azotobacter 

135 

35 

£.290 

21.0 

4S 

tt 

1T& 

30 

2.02C 

15.4 

43 

Sweet  clover 

144 

34 

2.230 

15.6 

50 

tt  tt 

141 

32 

1.S30 

« H 

51 

Garden  peas 

145 

30 

1. 820 

13.0 

52 

r n 

146 

33 

3.  l60 

23.3 

53 

Cowpeas 

1.49 

30 

2.150 

14.2 

54 

n 

150 

62 

> 

2.540 

13.6 

55 

Ca(NO  4-KpO 

J - *- 

44 

4.600 

47.0 

56 

tt 

50 

5.110 

55.0 

-IS- 


Oats  growing  in  the  presence  of  nodule  and  free  living  nitrogen-fixing  bacteria. 


-19- 

SmiAEY 

The  results  of  experiments  1 and  £ shc.v  that  there  is  no  disadvantage 
in  cross- inoculation  and  it  night  he  of  an  advantage,  as  shown  in  the  case  of 
the  performance  of  the  peanut  inoculation  in  experiment  1 and  the  vetch  in 
exp:  eri»i-ent2. 

The  results  of  experiment  3 show  that  the  oats  do  not  derive  any  benefit 
by  the  presence  of'  the  nodule  and  free- living  nitrogen-f ixing  b.  cteria.  The 
results  do  net  support  the  results  obtained  by  N.  V.  Joshi.  It  would  require 
ere  work  than  reported  herein  to  settle  either  one  of  these  questions. 

ACENOTOEDGIJMT 

The  author  takes  this  opportunity  to  express  his  gratitude  to  Professor 
A.  L.  Yhiting  under  whose  direction  and  careful  guidance  this  work  was  conducted. 


-20 


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